The use of triple resonance (TR) nuclear magnetic resonance (NMR) experiments for the resonance assignment of polypeptide chains via heteronuclear scalar connectivities (Montelione et al., J. Am Chem. Soc., 111:5474–5475 (1989); Montelione et al., J. Magn. Reson., 87:183–188 (1989); Kay et al., J. Magn. Reson., 89:496–514 (1990); Ikura et al., Biochemistry, 29:4659–8979 (1990); Edison et al., Methods Enzymol., 239:3–79 (1994)) is a standard approach which neatly complements the assignment protocol based on 1H—1H nuclear Overhauser effects (NOE) (Wüthrich, NMR of Proteins and Nucleic Acids, Wiley, New York (1986)). In addition, triple resonance NMR spectra are highly amenable to a fast automated analysis (Friedrichs et al., J. Biomol. NMR, 4:703–726 (1994); Zimmerman et al., J. Biomol. NMR, 4:241–256 (1994); Bartels et al., J. Biomol. NMR, 7:207–213 (1996); Morelle et al., J. Biomol. NMR, 5:154–160 (1995); Buchler et al., J. Magn. Reson., 125:34–42 (1997); Lukin et al., J. Biomol. NMR, 9:151–166 (1997)), yielding the 13Cα/β chemical shifts at an early stage of the assignment procedure. This enables both, the identification of regular secondary structure elements without reference to NOEs (Spera et al., J. Am. Chem. Soc., 113: 5490–5491 (1991)) and the derivation of (φ,ψ)-angle constraints which serve to reduce the number of cycles consisting of nuclear Overhauser enhancement spectroscopy (NOESY) peak assignment and structure calculation (Luginbühl et al., J. Magn. Reson., B 109:229–233 (1995)).
NMR assignments are prerequisite for NMR-based structural biology (Wüthrich, NMR of Proteins and Nucleic Acid, Wiley:New York (1986)) and, thus, for high-throughput (HTP) structure determination in structural genomics (Rost, Structure, 6:259–263 (1998); Montelione et al., Nature Struct. Biol., 6:11–12. (1999); Burley, Nature Struc Biol., 7:932–934 (2000)) and for exploring structure-activity relationships (SAR) by NMR for drug discovery (Shuker et al., Science, 274:1531–1534 (1996)). The aims of structural genomics are to (i) explore the naturally occurring “protein fold space” and (ii) contribute to the characterization of function through the assignment of atomic resolution three-dimensional (3D) structures to proteins. It is now generally acknowledged that NMR will play an important role in structural genomics (Montelione et al., Nature Struc. Biol., 7:982–984 (2000)). The resulting demand for HTP structure determination requires fast and automated NMR data collection and analysis protocols (Moseley et al., Curr. Opin. Struct. Biol., 9:635–642 (1999)).
The establishment of a HTP NMR structural genomics pipeline requires two key objectives in data collection. Firstly, the measurement time should be minimized in order to (i) lower the cost per structure and (ii) relax the constraint that NMR samples need to be stable over a long period of measurement time. The recent introduction of commercial cryogenic probes (Styles et al., J. Magn. Reson., 60:397–404 (1984); Flynn et al., J. Am Chem. Soc., 122:4823–4824 (2000)) promises to reduce measurement times by about a factor of ten or more, and will greatly impact the realization of this first objective. Secondly, reliable automated spectral analysis requires recording of a “redundant” set of multidimensional NMR experiments each affording good resolution (which requires appropriately long maximal evolution times in all indirect dimensions). Concomitantly, it is desirable to keep the total number of NMR spectra small in order to minimize “interspectral” variations of chemical shift measurements, which may impede automated spectral analysis. Straightforward consideration of this second objective would suggest increasing the dimensionality of the spectra, preferably by implementing a suite of four- or even higher-dimensional NMR experiments. Importantly, however, the joint realization of the first and second objectives is tightly limited by the rather large lower bounds of higher-dimensional TR NMR measurement times if appropriately long maximal evolution times are chosen.
Hence, “sampling limited” and “sensitivity limited” data collection regimes are distinguished, depending on whether the sampling of the indirect dimensions or the sensitivity of the multidimensional NMR experiments “per se” determines the minimally achievable measurement time. As a matter of fact, the ever increasing performance of NMR spectrometers will soon lead to the situation where, for many protein samples, the sensitivity of the NMR spectrometers do not constitute the prime bottleneck determining minimal measurement times. Instead, the minimal measurement times encountered for recording conventional higher-dimensional NMR schemes will be “sampling limited,” particularly as high sensitivity cryoprobes become generally available. As structure determinations of proteins rely on nearly complete assignment of chemical shifts, which are obtained using multidimensional 13C, 15N, 1H-TR NMR experiments (Montelione et al., J. Am Chem. Soc., 111:5474–5475 (1989); Montelione, et al., J. Magn. Reson., 87:183–188 (1989); Ikura et al., Biochemistry, 29:4659–8979 (1990)), the development of TR NMR techniques that avoid the sampling limited regime represents a key challenge for future biomolecular NMR methods development.
Reduced dimensionality (RD) TR NMR experiments (Szyperski et al., J. Biomol. NMR, 3:127–132 (1993); Szyperski et al., J. Am. Chem. Soc., 115:9307–9308 (1993); Szyperski et al., J. Magn. Reson., B 105:188–191 (1994); Brutscher et al., J. Magn. Reson., B 105:77–82 (1994); Szyperski et al., J. Magn. Reson., B 108: 197–203 (1995); Brutscher et al., J. Biomol. NMR, 5:202–206 (1995); Löhr et al., J. Biomol. NMR, 6:189–197 (1995); Szyperski et al., J. Am. Chem. Soc., 118:8146–8147 (1996); Szyperski et al., J. Magn. Reson., 28:228–232 (1997); Bracken et al., J. Biomol. NMR, 9:94–100 (1997); Sklenar et al., J. Magn. Reson., 130:119–124 (1998); Szyperski et al., J. Biomol. NMR, 11:387–405 (1998)), designed for simultaneous frequency labeling of two spin types in a single indirect dimension, offer a viable strategy to circumvent recording NMR spectra in a sampling limited fashion. RD NMR is based on a projection technique for reducing the spectral dimensionality of TR experiments: the chemical shifts of the projected dimension give rise to a cosine-modulation of the transfer amplitude, yielding peak doublets encoding n chemical shifts in a n−1 dimensional spectrum (Szyperski et al., J. Biomol. NMR, 3:127–132 (1993); Szyperski et al., J. Am. Chem. Soc., 115:9307–9308 (1993)). As a key result, this allows recording projected four-dimensional (4D) NMR experiments with maximal evolution times typically achieved in the corresponding conventional 3D NMR experiments (Szyperski et al., J. Biomol. NMR, 3:127–132 (1993); Szyperski et al., J. Am. Chem. Soc., 115:9307–9308 (1993); Szyperski et al., J. Magn. Reson. B 105:188–191 (1994); Szyperski et al., J. Magn. Reson., B 108: 197–203 (1995); Szyperski et al., J. Am. Chem. Soc., 118:8146–8147 (1996); Szyperski et al., J. Magn. Reson., 28:228–232 (1997); Bracken et al., J. Biomol. NMR, 9:94–100 (1997); Sklenar et al., J. Magn. Reson., 130:119–124 (1998); Szyperski et al., J. Biomol. NMR, 11:387–405 (1998)). Furthermore, axial coherences, arising from either incomplete insensitive nuclei enhanced by polarization transfer (INEPT) or heteronuclear magnetization, can be observed as peaks located at the center of the doublets (Szyperski et al., J. Am. Chem. Soc., 118:8146–8147 (1996)). This allows both the unambiguous assignment of multiple doublets with degenerate chemical shifts in the other dimensions and the identification of cross peak pairs by symmetrization of spectral strips about the position of the central peak (Szyperski et al., J. Am. Chem. Soc., 118:8146–8147 (1996); Szyperski et al., J. Biomol. NMR, 11:387–405 (1998)). Hence, observation of central peaks not only restores the dispersion of the parent, higher-dimensional experiment, but also provides access to reservoir of axial peak magnetization (Szyperski et al., J. Am. Chem. Soc., 118:8146–8147 (1996)). Historically, RD NMR experiments were first designed to simultaneously recruit both 1H and heteronuclear magnetization (Szyperski et al., J. Am. Chem. Soc., 118:8146–8147 (1996)) for signal detection, a feature that has also gained interest for improving transverse relaxation-optimized spectroscopy (TROSY) pulse schemes (Pervushin et al., Proc. Natl. Acad. Sci. USA, 94:12366–12371 (1997); Salzmann et al., J. Am. Chem. Soc., 121:844–848 (1999); Pervushin et al., J. Biomol. NMR, 12:345–348, (1998)). Moreover, RD two-spin coherence NMR spectroscopy (Szyperski et al., J. Biomol. NMR, 3:127–132 (1993)) subsequently also called zero-quantum/double-quantum (ZQ/DQ) NMR spectroscopy (Rexroth et al., J. Am. Chem. Soc., 17:10389–10390 (1995)), served as a valuable radio-frequency (r.f.) pulse module for measurement of scalar coupling constants (Rexroth et al., J. Am. Chem. Soc., 17: 10389–10390 (1995)) and cross-correlated heteronuclear relaxation (Reif et al., Science, 276:1230–1233 (1997); Yang et al., J. Am. Chem. Soc., 121:3555–3556 (1999); Chiarparin et al., J. Am. Chem. Soc., 122:1758–1761 (2000); Brutscher et al., J. Magn. Reson., 130:346–351 (1998); Brutscher, Concepts Magn. Reson., 122:207–229 (2000)).
The present invention is directed to overcoming the deficiencies in the art.